Antigen-specific t cells for inducing immune tolerance

ABSTRACT

Described herein are agents and methods for targeting antigen-specific B cells using engineered T cells, such as regulatory T cells or cytotoxic T cells, or bi-specific antibodies. The agents and methods can be used to reduce undesirable immune responses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/780,584, which is a 35 U.S.C. § 371 U.S. national phase applicationof International Appl. No. PCT/US2016/055656, having an internationalfiling date of Oct. 6, 2016, claiming the benefit under 35 U.S.C. §119(e) to U.S. Appl. No. 62/263,108, filed Dec. 4, 2015, U.S. Appl. No.62/359,886, filed Jul. 8, 2016, and U.S. Appl. No. 62/393,245, filedSep. 12, 2016. The contents of these applications are incorporated byreference herein in their entireties.

GOVERNMENT RIGHTS

This invention was made with government support under R21 HL127495, R01HL126727 and R01 HL061833 awarded by the National Institutes of Health.The government has certain rights in the invention.

FIELD

Described herein are agents and methods for targeting antigen-specific Bcells using engineered T cells, such as cytotoxic T cells or regulatoryT cells, or bispecific antibodies. The agents and methods can be used toinduce immune tolerance, e.g., to reduce undesirable immune responses.

BACKGROUND

Many therapies can induce undesired immune responses, such as antibodyresponses to therapeutic proteins or allergens or toxins used asimmunotoxins. Undesired immune responses also may arise in the contextof transplant rejection, allergies, allergic responses, and asthma.Thus, it may be desirable to suppress immune responses to therapeuticagents, allograft transplants, and allergens.

Methods for immunosuppression have been disclosed that involve expandingnon-specific regulatory T cells. However, non-specific regulatory Tcells can suppress desired immune responses, such as responses topathogenic infections and cancer. Thus, there remains a need for a moretargeted approach to immunosuppression.

One example of a potentially immunogenic therapeutic agent is clottingFactor VIII (FVIII), used to treat hemophilia. Hemophilia is the secondmost common congenital bleeding disorder and is characterized byfrequent bleeds at joint levels resulting in cartilage fibrosis, loss ofjoint space, and debilitation. Hemophilia affects the knees, ankles,hips, shoulders, elbows and bleeding into closed spaces can be fatal.Hemophilia A is caused by a genetic deficiency in clotting factor VIII,an essential blood-clotting protein. Depending on the mutation,hemophilia A patients lack all or part of the FVIII protein. Currently,treatment methods for hemophilia consist of infusions of eitherrecombinant or plasma-derived clotting factor concentrates, usuallyadministered in response to bleeds. However, greater than 20% ofhemophilia A patients exhibit undesired immune responses against theseprotein therapies, and make antibodies against therapeutic FVIIIproteins that undermine the treatment and inhibit clotting. ConventionalFVIII therapies also require frequent injection/infusion over thepatient's lifetime, and are associated with very high costs.

Thus, there exists a need for effective approaches to reducingundesirable immune responses, such as undesirable immune responses totherapeutic agents, allograft transplants, and allergens.

SUMMARY

Described herein are agents and methods for targeting antigen-specific Bcells using engineered T cells, such as regulatory T cells of cytotoxicT cells, or bispecific antibodies. The agents and methods can be used toreduce undesirable immune responses, as discussed in more detail below.

In accordance with some embodiments, there are provided methods oftargeting antigen-specific B cells, comprising exposing B cells specificto a target antigen to regulatory T cells that express on their cellsurface the target antigen or a domain thereof recognized by theantigen-specific B cells. Also provided are methods of targetingantigen-specific B cells, comprising exposing B cells specific to atarget antigen to (i) regulatory T cells that express on their cellsurface a single chain antibody specific for the target antigen, and(ii) the target antigen. In some embodiments, the single chain antibodyis bound to the target antigen or domain thereof recognized by theantigen specific B cells before exposure to the antigen specific Bcells. Also provided are methods of targeting antigen-specific B cells,comprising exposing B cells specific to a target antigen to regulatory Tcells bound by a bi-specific antibody comprising a T cell-binding endand a B cell-targeting end, wherein the B cell-targeting end comprises adomain of the target antigen recognized by the antigen-specific B cells.In any of these embodiments, the regulatory T cells may be selected fromCD4⁺CD25^(hi)CD127^(lo)Foxp3⁺ regulatory T cells,CD4⁺CD25^(hi)CD127^(lo)Foxp3⁺ Helios⁺ regulatory T cells,CD4⁺CD25^(hi)Foxp3⁺ regulatory T cells, and CD4⁺CD25^(hi)Foxp3⁺Helios⁺regulatory T cells.

In accordance with some embodiments, there are provided methods oftargeting antigen-specific B cells, comprising exposing B cells specificto a target antigen to cytotoxic T cells that express on their cellsurface the target antigen or a domain thereof recognized by theantigen-specific B cells. Also provided are methods of targetingantigen-specific B cells, comprising exposing B cells specific to atarget antigen to (i) cytotoxic T cells that express on their cellsurface a single chain antibody specific for the target antigen and (ii)the target antigen. In some embodiments, the single chain antibody isbound to the target antigen or domain thereof recognized by the antigenspecific B cells before exposure to the antigen specific B cells. Alsoprovided are methods of targeting antigen-specific B cells, comprisingexposing B cells specific to a target antigen to cytotoxic T cells boundby a bi-specific antibody comprising a T cell-binding end and a Bcell-targeting end, wherein the B cell-targeting end comprises a domainof the target antigen recognized by the antigen-specific B cells. Inaccordance with any of these methods, the cytotoxic T cell may beselected from CD8+ T cells and natural killer (NK) T cells.

In accordance with any of the methods, the cytotoxic T cells orregulatory T cells may be transduced with a B-cell-targeting antibodyreceptor (BAR) construct comprising (i) an extracellular domaincomprising the target antigen or domain thereof and (ii) anintracellular signaling domain. In accordance with any of these methods,the intracellular signaling domain may comprise one or more signalingdomains, such as one or more signaling domains from CD28-CD3ζ 4-1BB,ICOS, and CTLA-4.

In accordance with any of these methods, the target antigen may be atherapeutic agent, such as a therapeutic protein or an allergen or toxinused as an immunotoxin. In some embodiments, the target antigen isassociated with an autoimmune disorder, such as multiple sclerosis,diabetes, uveitis, thyroiditis, myasthenia gravis, antiphospholipidsyndrome (APS), or an undesired immune response to a therapeutic agent,such as a biotherapeutic agent used to treat a genetic disease, such ashemophelia or Pompe's, or an allergen or antigen used as an immunotoxin,or may be associated with an allergy, allergic response or asthma, ormay be an antigen of an allograft transplant. In some embodiments, thetarget antigen is a therapeutic protein selected from human clottingfactor VIII, the C2 domain of human clotting factor VIII, the A-2 domainof human clotting factor VIII, and the A2-C2 domain of human clottingfactor VIII, human clotting factor IX, myelin basic protein (MBP) orother antigens associated with multiple sclerosis, an antigen associatedwith diabetes, an antigen associated with uveitis, an antigen associatedwith thyroiditis, an antigen associated with myasthenia gravis, anantigen associated with antiphospholipid syndrome (APS), an antigen ofan allograft transplant, such as a cell, tissue, or organ of anallograft transplant, or an antigen associated with an allergy orallergic response, or asthma.

In accordance with any of these methods, the method may be effected in apatient suffering from or at risk of developing an undesirable immuneresponse to the target antigen. In some embodiments, the patient issuffering from an autoimmune disorder, such as one or more selected fromthe multiple sclerosis, diabetes, and uveitis, and/or is receiving abiotherapeutic treatment for a genetic disease, such as Pompe's,hemophilia (including hemophilia A and hemophilia B), thyroiditis, ormyasthenia gravis, and/or is receiving an allograft transplant. Inspecific embodiments, the patient is suffering from or at risk ofdeveloping an undesired immune response to Factor VIII therapy. In someembodiments, the method is effective to reduce or prevent the patient'simmune response to the target antigen.

Also provided are regulatory T cells that express on their cell surfacea target antigen or a domain thereof recognized by an antigen-specific Bcell. In accordance with any of these embodiments, the regulatory T cellmay be transduced with a B-cell-targeting antibody receptor (BAR)construct comprising (i) an extracellular domain comprising the targetantigen or domain thereof and (ii) an intracellular signaling domain. Insome embodiments, the intracellular signaling domain comprises one ormore signaling domains, such as one or more signaling domains fromCD28-CD3ζ, 4-1BB, ICOS, and CTLA-4. Also provided are regulatory T cellsthat expresses on their surface a single chain antibody specific for atarget antigen. Also provided are regulatory T cells bound by abi-specific antibody comprising a T cell-binding end and a Bcell-targeting end, wherein the B cell-targeting end comprises a domainof a target antigen recognized by an antigen-specific B cell. Inaccordance with any of these embodiments, the regulatory T cells may beisolated using CD4, CD25, CD127 cell surface markers. In any of theseembodiments, the regulatory T cells may be selected fromCD4⁺CD25^(hi)CD127^(lo)Foxp3⁺ regulatory T cells,CD4⁺CD25^(hi)CD127^(lo)Foxp3⁺ Helios⁺ regulatory T cells,CD4⁺CD25^(hi)Foxp3⁺ regulatory T cells, and CD⁺CD25^(hi)Foxp3⁺Helios⁺regulatory T cells.

Also provided are cytotoxic T cells that express on their cell surface atarget antigen or a domain thereof recognized by an antigen-specific Bcell. In accordance with any of these embodiments, the cytotoxic T cellmay be transduced with a B-cell-targeting antibody receptor (BAR)construct comprising (i) an extracellular domain comprising the targetantigen or domain thereof and (ii) an intracellular signaling domain. Insome embodiments, the intracellular signaling domain comprises one ormore signaling domains, such as one or more signaling domains fromCD28-CD3ζ, 4-1BB, and ICOS. Also provided are cytotoxic T cells thatexpresses on their cell surface a single chain antibody specific for atarget antigen. Also provided are cytotoxic T cells bound by abi-specific antibody comprising a T cell-binding end and a Bcell-targeting end, wherein the B cell-targeting end comprises a domainof a target antigen recognized by an antigen-specific B cell. Inaccordance with any of these embodiments, the cytotoxic T cell may beselected from CD8+ T cells and natural killer (NK) T cells.

In accordance with any of these embodiments, the target antigen may beany one or more as discussed above with regard to methods.

Also provided are regulatory T cells or cytotoxic T cells as describedherein, for use in reducing undesired immune responses, inducing immunetolerance, treating autoimmune diseases or disorders, or enhancing apatient's response to a biotherapeutic treatment for a genetic disease.

Also provided are uses of regulatory T cells or cytotoxic T cells asdescribed herein in the preparation of medicaments for reducingundesirable immune responses, inducing immune tolerance, treatingautoimmune diseases or disorders, or enhancing a patient's response to abiotherapeutic treatment for a genetic disease.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. Overview of methods of targeting antigen -specific B cells byengineered CD8+ T cells as described herein. On the left, a CD8+ T cellengineered to express a target antigen domain (such as FVIII C2 and A2)binds to B cell IgM receptors on the surface of an FVIII-specific Bcell. On the right, a CD8+ T cell engineered to express a single chainantibody specific for a target antigen (such as FVIII) bind to thetarget antigen (such as FVIII) which in turn is bound to anFVIII-specific B cell. In both approaches, the T cells induce killing ofthe antigen-specific B cells.

FIG. 2. Schematic of bi-specific antibodies comprising a T cell-bindingend and a B cell-targeting end, wherein the B cell-targeting endcomprises a domain of a target antigen recognized by an antigen-specificB cell. The target antigen domain (such as FVIII domains) is cloned intoone IgG arm, and the T cell-binding moiety (e.g., anti-CD8 or NK marker)is cloned into the other arm. When the targeted antigen-specific B cellsbind the antigen epitope of the bispecific antibody, the antibody-boundT cells induce killing of the antigen-specific B cells.

FIG. 3. Schematic illustration of construct used to make a FVIII-C2 Bcell antibody receptor (BAR) construct. The genes (from N to C terminus)are: mouse CD4 N-terminal signaling peptide, FVIII-C2, human CD28transmembrane, intracellular, human CD3ζ, an internal ribosomal entrysite (IRES) and GFP sequence. The IRES allows the BAR construct and GFPto be expressed as a single mRNA molecule, so that measurement of GFPcorrelates to BAR construct expression. A control constructing havingthe gene encoding OVA in place of the FVIII-C2 gene also is depicted(OVA-BAR construct).

FIG. 4. Expression of BAR in CD8 T cells. Isolated CD8+ T cells wereactivated and transduced with replication deficient BAR retrovirus usingretronectin. 24 hrs post transduction, GFP expression can be visualizedin both FVIII-C2 and OVA BAR transduced T cells.

FIG. 5. Expression of BAR on T cell surface. The surface expression ofthe FVIII-C2 domain and OVA on the T cells is measured by flow cytometryusing known antibodies against FVIII-C2 and OVA. FVIII-A2 antibody andanti-OVA were used as control.

FIG. 6. Proliferation of BAR Transduced T cells. To observe theproliferation competency of the BAR expressing T cells, T cells weretransduced and rested for 10 days, irradiated-PBMC pulsed withanti-FVIII sera were added at 2:1 to BAR expressing T cells. Cells weregrown for 48 hrs and radioactive titrated thymidine uptake assay wasperformed for 24 hrs. The thymidine uptake by the proliferating T cellswas measured. The figures show an increase in proliferation of C2-BAR Tcells proliferation when anti-FVIII sera was used compared to thecontrols.

FIG. 7. FVIII-C2 BAR transduced CD8+ T cells are able to kill α-FVIIIhybridoma, as shown by JAM test. 2C11 (anti-C2) B cell hybridoma cellswere radiolabeled with titrated thymidine for 24 hrs and C2-BARtransduced CD8 T cells were added at various ratios and incubated for 5hrs. At the end of the test, radioactivity was measured. The data showsa decrease in radiolabeled cells compare to that of ova controls, whichcorresponds to reduced viability of B cell hybridoma cells.

FIG. 8. FVIII-C2 BAR transduced CD8 T cells kill α-FVIII hybridoma shownby ELISPOT assay. 2C11 (anti-C2) B cell hybridoma cells were incubatedwith C2-BAR CD8 T cells at various ratios for 5 hrs and are then grownfor 12hrs on a FVIII coated plate. Number of spots formed at the end ofthe assay was quantified, a decrease in number of spots formed per well(correlates to number of viable antibody secreting B cells) in C2-BARtransduced CD8 T cells was detected compared to control.

FIG. 9. Cumulative results of 3 individual ELISPOT performed under thesame conditions.

FIG. 10. FVIII-C2 BAR transduced CD8 T cells kill α-FVIII hybridoma, asshown by ELISPOT assay. 3G6 B cell hybridoma cells were incubated withC2-BAR transduced CD8 T cells at various ratios for 5hrs and are thengrown for 12hrs on a FVIII coated plate. Number of spots formed at theend of the assay was quantified, the authors observed a decrease innumber of spots formed per well (correlates to number of viable antibodysecreting B cells) in C2-BAR transduced CD8 T cells compared to control.

FIG. 11. Image of A2-BAR mediated cytolysis of α-FVIII hybridoma. A2-BARtransduced CD8 T cells are incubated with α-FVIII hybridoma. Images wastaken at 5, 20, 30, 40 and 55 minutes

FIGS. 12A-12B. Schematic diagram of chimeric antigen receptor (CAR) andB-cell-targeting antibody receptor (BAR). FIG. 12A illustrates CAR,wherein the extracellular domain is a single chain variable fragment(ScFv) of an antibody that recognizes a target antigen. FIG. 12Billustrates BAR, wherein the extracellular domain is a target antigen ordomain thereof recognized by a B cell or antibody. The transmembrane andintracellular signaling domains of the illustrated CAR and BARconstructs comprise CD28-CDζ3.

FIGS. 13A-13C. Generation and expression of BAR on human CD4+ T cells.FIG. 13A is a schematic illustration of BAR constructs. The genes (fromN to C terminus) are: signaling peptide, FVIII-C2 or FVIII-A2, humanCD28-CD3ζ transmembrane/intracellular domains. A control constructhaving the gene encoding ovalbumin (OVA) also is depicted (“OVA BAR”).FIG. 3B illustrates an expression cassette of a retroviral vectorencoding a BAR construct with a green fluorescent protein (GFP) reporterunder the control of internal ribosomal entry site (IRES). The IRESallows the BAR construct and GFP to be expressed as a single mRNAmolecule, so that measurement of GFP correlates to BAR constructexpression. FIG. 13C shows the flow cytometry analysis of surfaceexpression of C2 and OVA in transduced human CD4+ T cells by stainingwith monoclonal anti-C2 and polyclonal rabbit anti-OVA IgG,respectively, and demonstrates that BARs were successfully expressed ontransduced cells.

FIG. 14. Proliferation response of human CD4+ T cells expressing OVA-BARto anti-OVA antibody. Human CD4+ T cells were FACS sorted from PBMC andtransduced to express OVA-BAR using concentrated retroviral supernatant.Transduced cells were labeled with cell proliferation dye eFluor 450 andcultured with either anti-OVA IgG or control IgG for 3 days, in thepresence of irradiated PBMC. Cell proliferation was analyzed by flowcytometry. The cells were gated on live GFP+ cells. This figure showsthat OVA-BAR transduced T cells proliferated specific to anti-OVA IgGbut not to control IgG, which confirms that the BAR expressed on the Tcells is functional.

FIG. 15. FACS analysis of the quality of BAR-transduced regulatory Tcells (Tregs) after about 3 weeks in vitro expansion. This figure showsthat BAR-transduced, long term in vitro-expanded human Tregs retainedthe xo-expression of Foxp3 and Helios, which are markers for functionalhuman Tregs.

FIGS. 16A-16C. Suppression of FVIII-specific B cell activity in axenogeneic mouse model in vivo. FIG. 16A shows a timeline for generationof BAR-transduced and in vitro-expanded human regulatory T cells(Tregs). FIG. 16B outlines the experimental protocol. FIG. 16C showsanti-FVIII antibody levels measured by ELISA assay with a standard curvegenerated using a mixture of anti-A2 and anti-C2 monoclonal antibodies.(*p<0.05, student t test, single tail distribution.) This figure showsthat adoptive transfer of the mixture containing equal number of A2 BARtransduced human Tregs and C2 BAR transduced human Tregs ((A2+C2) BARhTregs) prevented anti-FVIII antibody development in all 5 mice in thegroup, which confirmed the effectiveness of BAR-Treg therapy fortreating unwanted immune responses.

FIG. 17. Schematic diagram of chimeric antigen receptor (CAR) andB-cell-targeting antibody receptor (BAR) constructs. On the left (CAR),a T cell engineered to express a single chain antibody specific for atarget antigen (such as FVIII) is bound to target antigen (such asFVIII) which in turn is bound to an FVIII-specific B cell. On the right(BAR), a T cell is engineered to express a target antigen domain (suchas FVIII C2) which is recognized by and bound to a FVIII-specific Bcell.

FIGS. 18A-18B. Proposed mechanism of action for the ability ofengineered regulatory T cells as described herein to suppress B cellresponses. FIG. 18A shows that regulatory T cells may directly suppressB cells. FIG. 18B shows that engineered regulatory T cells mayindirectly suppress B cell via effector T cells.

FIG. 19. Comparison of cell populations before FACS sorting and afterFACS sorting. As shown, the CD4⁺CD25^(hi)CD127^(lo) regulatory T cellsalso express Foxp3 and Helios.

FIG. 20. Illustration of FACS sorting of regulatory T cells to obtainregulatory T cells having a CD4⁺CD25^(hi)CD127^(lo)Foxp3⁺ Helios⁺phenotype. As shown, the CD4⁺CD25^(hi)CD127^(lo) cells also expressFoxp3 and Helios.

DETAILED DESCRIPTION

Described herein are agents and methods for targeting antigen-specific Bcells using engineered regulatory T cells or cytotoxic T cells andbispecific antibodies. The agents include engineered human cytotoxic Tcells (such as CD8+ or NK T cells), engineered human regulatory T cells,and bi-specific antibodies. The agents and methods can be used to reduceundesirable immune responses, including minimizing or eliminatingadverse, undesirable immune responses, such as may arise in response toprotein therapy for genetic diseases, or to allergens or toxins used inimmunotoxins. The approaches described herein use the engineeredregulatory T cells, or bispecific antibodies to suppress or killantigen-specific B cells that are the precursors for antibody productionagainst the target antigen (e.g., therapeutic proteins or allografts),thus reducing or eliminating the undesired antibody response.

Definitions

Technical and scientific terms used herein have the meanings commonlyunderstood by one of ordinary skill in the art to which the presentinvention pertains, unless otherwise defined. Reference is made hereinto various methodologies known to those of ordinary skill in the art.Publications and other materials setting forth such known methodologiesto which reference is made are incorporated herein by reference in theirentireties as though set forth in full. Any suitable materials and/ormethods known to those of ordinary skill in the art can be utilized incarrying out the present invention. However, specific materials andmethods are described. Materials, reagents and the like to whichreference is made in the following description and examples areobtainable from commercial sources, unless otherwise noted.

As used herein, the singular forms “a,” “an,” and “the” designate boththe singular and the plural, unless expressly stated to designate thesingular only.

The term “about” and the use of ranges in general, whether or notqualified by the term about, means that the number comprehended is notlimited to the exact number set forth herein, and is intended to referto ranges substantially within the quoted range while not departing fromthe scope of the invention. As used herein, “about” will be understoodby persons of ordinary skill in the art and will vary to some extent onthe context in which it is used. If there are uses of the term which arenot clear to persons of ordinary skill in the art given the context inwhich it is used, “about” will mean up to plus or minus 10% of theparticular term.

As used herein “subject” denotes any mammal, including humans.

Cytotoxic T cells and natural killer (NK) T cells play an important rolefor immune defense against intracellular pathogens, including virusesand bacteria, and for tumor surveillance. Cytotoxic T cells recognizesurface markers on other cells in the body and label those cells fordestruction. The most well-understood cytotoxic T cells are those thatexpress CD8 (CD8⁺ T cells), and NK T cells.

Regulatory T cells suppress immune responses of other cells. They comein several forms with the most well-understood being those that expressCD4, CD25, Foxp3, and Helios (CD4⁺CD25⁺ Tregs). These cells are involvedin shutting down immune responses after they have successfullyeliminated invading organisms, and in preventing autoimmunity.

Previously, T cells have been rendered specific for cancer antigens(CARs) in order to specifically destroy cancer cells, e.g., in order topromote an immune response. However, the approaches described herein useengineered cytotoxic T cells or engineered regulatory T cells in orderto modulate (turn off) undesirable immune responses, e.g., to reduce animmune response. The use of engineered cytotoxic T cells, engineeredregulatory T cells, or bispecific antibodies described herein tospecifically target antigen-specific B cells for killing has notheretofore been described.

In accordance with some embodiments, a regulatory T cell or cytotoxic Tcell is engineered to express on its cell surface a target antigen or adomain thereof recognized by an antigen-specific B cell. Such engineeredregulatory T cells or cytotoxic T cells can be used in methods oftargeting antigen-specific B cells, comprising exposing B cells specificto a target antigen to regulatory T cells or cytotoxic T cells thatexpress on their cell surface the target antigen or a domain thereofrecognized by the antigen-specific B cells. In accordance with suchmethods, the engineered regulatory T cells or engineered cytotoxic Tcells are brought into proximity/contact with the antigen-specific Bcells via the target antigen or domain thereof expressed on the surfaceof the T cells and bound by the antigen-specific B cells, such that theengineered T cells induce killing of the antigen-specific B cells.

In accordance with other embodiments, a regulatory T cell or cytotoxic Tcell is engineered to express on its cell surface a single chainantibody that is specific for a target antigen. Such engineeredregulatory T cells or cytotoxic T cells can be used in methods oftargeting antigen-specific B cells, comprising exposing B cells specificto a target antigen to (i) regulatory T cells or cytotoxic T cells thatexpress on their cell surface a single chain antibody specific for thetarget antigen and (ii) the target antigen (or a fragment thereof thatcan be bound by both the single chain antibody and antigen-specific Bcells). In some embodiments, the single chain antibody is bound to thetarget antigen or domain thereof recognized by the antigen specific Bcells before exposure to the antigen specific B cells.

In accordance with other embodiments, a bispecific antibody is preparedthat has a T cell-binding end and a B cell-targeting end, wherein the Bcell-targeting end comprises a domain of the target antigen recognizedby the antigen-specific B cells cloned into one IgG arm, and a Tcell-binding moiety cloned into the other arm. In some embodiments, theT cell-binding end comprises an anti-CD8 single chain antibody or ananti-CD3e single chain antibody. In other embodiments, the Tcell-binding end comprises an antibody that specifically recognizes theregulatory T cell. The bispecific antibodies are useful in methodscomprising exposing antigen-specific B cells to regulatory T cells orcytotoxic T cells bound by the bispecific antibodies. In such methods,the regulatory T cells or cytotoxic T cells are brought intoproximity/contact with the antigen-specific B cells via binding betweenthe bispecific antibody and the T cells and antigen-specific B cells,such that the regulatory T cells or cytotoxic T cells induce suppressionor killing of the antigen-specific B cells.

Cytotoxic T cells useful herein may be selected from CD8+ T cells andnatural killer (NK) T cells. In accordance with any of theseembodiments, the cytotoxic T cell may maintain its phenotype aftertransduction and long-term expansion.

Regulatory T cells useful herein may be isolated by FluorescentActivated Cell Sorting (FACS) based on the cell surface markers, such asCD4, CD 25, and CD127. In some embodiments, regulatory T cells ofinterest include those with little or no expression of CD127)(CD127^(lo)), and/or with high expression of CD25 (CD25^(hi)). In someembodiments, the regulatory T cells express CD4, CD25, Foxp3, andHelios. In some embodiments, the regulatory T cells areCD4⁺CD25^(hi)CD127^(lo)Foxp3⁺Helios⁺ regulatory T cells (FIG. 8, FIG.9). In other embodiments, the regulatory T cells areCD4⁺CD25^(hi)CD127^(lo)Foxp3⁺ regulatory T cells, CD4⁺CD25^(hi)Foxp3⁺regulatory T cells or CD4⁺CD25^(hi)Foxp3⁺Helios⁺ regulatory T cells. Inaccordance with any of these embodiments, the regulatory T cell maymaintain its phenotype after transduction and long-term expansion.

In accordance with any of these embodiments, autologous polyclonalregulatory T cells and HLA-matched allogeneic regulatory T cells can beused as donor cells. In some embodiments, endogenous TCR on donorregulatory T cells can be inactivated using existing technologies suchas CRISPR/Cas9.

In accordance with any of these embodiments, autologous polyclonalcytotoxic T cells and HLA-matched allogeneic cytotoxic T cells can beused as donor cells. In some embodiments, endogenous TCR on donorcytotoxic T cells can be inactivated using existing technologies such asCRISPR/Cas9.

Engineered regulatory T cells or cytotoxic T cells as described hereincan be made by adapting methods known in the art, including the methodsof isolating and transducing T cells described in U.S. ProvisionalApplication 61/821,857 and U.S. patent application Ser. No. 14/889,962,filed Nov. 9, 2015 (published as US 2016/0194605), which areincorporated herein by reference in their entireties. Gene and proteinsequences of target antigens and domains thereof recognized byantigen-specific B cells are known in the art, and include those setforth in U.S. Provisional Application 61/821,857 and U.S. applicationSer. No. 14/889,962 and in U.S. Provisional Application 61/990,456 andPCT Application PCT/US15/29642, filed May 7, 2015 (published as WO2015/171863), which are incorporated herein by reference in theirentireties.

In accordance with any of these embodiments, the cytotoxic T cells orregulatory T cells may be transduced with a B-cell-targeting antibodyreceptor (BAR) construct comprising (i) an extracellular domaincomprising the target antigen or a domain thereof recognized by the Bcell and (ii) an intracellular signaling domain. For cytotoxic T cells,the intracellular signaling domain may comprise one or more signalingdomains, such as one or more signaling domains of CD28-CD3ζ, 4-1BB, andICOS. For regulatory T cells, the intracellular signaling domain maycomprise one or more signaling domains, such as one or more signalingdomains of CD28-CD3ζ, 4-1BB, ICOS, and CTLA-4.

In accordance with any of these embodiments, the target antigen ordomain thereof recognized by the antigen-specific B cells may be atherapeutic agent, such as a therapeutic protein or an allergen ortoxins used as an immunotoxin, such as a therapeutic protein selectedfrom human clotting factor VIII, human clotting factor IX, the C2 domainof human clotting factor VIII, the A2 domain of human clotting factorVIII, and the A2-C2 domain of human clotting factor VIII, an antigenassociated with diabetes, such as insulin or glutamic acid decarboxylase(GAD65), an antigen associated with uveitis, such as arrestin, myelinbasic protein (MBP) or other antigens associated with multiplesclerosis, such as thyroperoxidase (TPO), an antigen associated withthyroiditis, such as thyroperoxidase (TPO), an antigen associated withmyasthenia gravis, such as acetylcholine receptor (AchR), an antigenassociated with antiphospholipid syndrome (APS) (as may be associatedwith systemic lupus erythematosus (“SLE” or “Lupus”) or repeatedmiscarriage), or a domain of any of the foregoing recognized by the Bcell. Alternatively, the target antigen may be associated with atransplant, such as a cell, tissue, or organ of an allograft transplant.Alternatively, the target antigen may be an antigen associated with anallergy, allergic response, or asthma.

Any of the engineered T cells or antibodies described herein can be usedin any of the methods outlined above. In some embodiments, the method iseffected in a patient suffering from or at risk of developing anundesirable immune response to the target antigen. In some embodiments,the patient is suffering from autoimmune diseases, anti-drug antibodydevelopment, or allergy. In some embodiments, the patient is sufferingfrom an autoimmune disorder, such as one or more selected from the groupconsisting of multiple sclerosis, diabetes, uveitis, thyroiditis,myasthenia gravis, or antiphospholipid syndrome (APS), or is receiving abiotherapeutic treatment for a genetic disease, such as Pompe's orhemophilia (including hemophilia A and hemophilia B). In specificembodiments, the patient is a hemophilia patient (including hemophilia Aand hemophilia B). In other specific embodiments, the patient isreceiving a transplant. In other specific embodiments, the patient issuffering from or at risk of developing an undesired immune response toFactor VIII therapy. In accordance with any of these embodiments, themethod may be effective to reduce or prevent the patient's immuneresponse to the target antigen. In other specific embodiments, thepatient suffers from an allergy or allergic response or asthma, andtarget antigen may be an antigen associated with the allergy or allergicresponse or asthmatic response, and the method may be effective toreduce or eliminate the allergy or allergic response or to treat theasthma or reduce the asthmatic response.

In accordance with any of the regulatory T cell embodiments, the targetantigen is not desmoglein (Dsg) 3 or a domain thereof recognized byantigen-specific B cells. In some embodiments, the target antigen is notDsg3 EC1, EC2, EC3, or EC4. In accordance with any of the cytotoxic Tcell embodiments, the target antigen is not desmoglein (Dsg) 3 or adomain thereof recognized by antigen-specific B cells. In someembodiments, the target antigen is not Dsg3 EC1, EC2, EC3, or EC4.

EXAMPLES

T cell isolation, activation, transduction, flow-cytometry, cellproliferation assays, JAM assays, and ELISPOT assays were conductedaccording to procedures known in the art, and can be conducted inmanners similar to those described in U.S. Provisional Application61/821,857, U.S. Provisional Application 61/990,456, U.S. applicationSer. No. 14/889,962, filed Nov. 9, 2015 (published as US 2016/0194605),and PCT Application PCT/US15/29642, filed May 7, 2015 (published asWO2015/171863), which are incorporated herein by reference in theirentireties.

Example 1—Construction of FVIII-C2 B Cell Antibody Receptor (BAR)Construct

A mouse CD4 N-terminal signaling peptide, FVIII-C2,human CD28transmembrane, intracellular, human CD3ζ, an internal ribosomal entrysite (IRES) and GFP gene were cloned into a vector to create theFVIII-C2 BAR construct. A control construct where the gene encoding OVAreplaced FVIII-C2 also was prepared (the OVA-BAR construct). The IRESallows the BAR construct and GFP to be expressed as a single mRNAmolecule, such that measurement of GFP correlates to BAR constructexpression. The results in FIG. 3 show that the genes were successfullycloned into the vector.

This BAR construct can be used to transduce cytotoxic T cells orregulatory T cells.

Example 2—Expression of BAR on the Surface of Cytotoxic T Cells

Isolated CD8+application Ser. No. cytotoxic T cells were activated andtransduced with replication deficient BAR retrovirus using retronectin.GFP expression was analyzed 24 hrs post transduction. FIG. 4 shows thatGFP expression can be visualized in both FVIII-C2 BAR and OVA transducedT cells, which shows that both constructs were efficiently transducedinto T cells.

Flow cytometry using known antibodies against FVIII-C2 and OVA was usedto determine whether the BAR construct was expressed on the T cellsurface. Anti-A2 and anti-OVA antibodies were used as controls. Theresults in FIG. 5 show that the C2-BAR construct was expressed on the Tcell surface.

To observe the proliferation competency of the BAR-expressing T cells, Tcells were transduced and rested for 10 days, and then irradiated-PBMCpulsed with anti-FVIII sera were added at 2:1 to BAR-expressing T cells.Cells were grown for 48 hrs and radioactive tritiated thymidine uptakeassay was performed for 24 hrs. The thymidine uptake by theproliferating T cells was measured. The results in FIG. 6 show anincrease in proliferation of C2-BAR T cells proliferation whenanti-FVIII sera was used, as compared to the controls.

Example 3—Killing of B Cells by BAR-Transduced Cytotoxic T Cells

To determine whether FVIII-C2 BAR transduced cytotoxic T cells are ableto kill α-FVIII hybridoma, a JAM test was conducted with 2C11 (anti-C2)B cell hybridoma cells. The 2C11 (anti-C2) B cell hybridoma cells wereradiolabeled with tritiated thymidine for 24 hrs and C2-BAR transducedCD8+ T cells were added at various ratios and incubated for 5 hrs. Atthe end of the test, radioactivity was measured. As shown in FIG. 7, adecrease in radiolabeled hybridoma cells, which corresponds to reducedviability of B cell hybridoma cells, was detected compare to the control(OVA transduced T cell). The results show that FVIII-C2 BAR transduced Tcells are able to kill B cell hybridoma cells.

To quantify the hybridoma cell viability, an ELISPOT assay was carriedout with FVIII-C2 BAR transduced CD8+ T cells. 2C11 (anti-C2) B cellhybridoma cells were incubated with C2-BAR CD8 T cells at various ratiosfor 5 hrs and are then grown for 12 hrs on a FVIII coated plate. Numberof spots formed at the end of the assay was quantified. The results inFIGS. 8-10 show a decrease in number of spots formed per well (whichcorrelates to the number of viable antibody secreting B cells) in C2-BARtransduced CD8+ T cells compared to that of OVA control.

To observe A2-BAR mediated cytolysis of α-FVIII hybridoma, FVIII A2-BARtransduced CD8 T cells were incubated with α-FVIII hybridoma. Imageswere taken at 5, 20, 30, 40 and 55 minutes after the incubation. Asshown in FIG. 11, cytolysis of the hybridoma by FVIII A2-BAR transducedCD8 T cells was observed.

Example 4—Construction, Expression of BAR on Human CD4+ Regulatory TCells

Constructs as depicted in FIG. 13A and FIG. 13B were prepared. Inparticular, constructs comprising a signaling peptide, FVIII-C2 orFVIII-A2, human CD28-CD3ζ transmembrane/intracellular domains wereprepared. As control, a OVA-BAR construct was prepared as illustrated inthe figure. A retroviral vector, pRetro-IRES-Zsgreen was used to expressthe BAR constructs. The GFP reporter gene under the control of the IRESelement enables tracking of successfully transduced cells. To confirmthe proper expression of BAR, human CD4+ regulatory T cells wereretrovirally transduced with either C2 BAR or OVA BAR. Flow cytometryusing known antibodies against FVIII-C2 and OVA was used to determinewhether the BAR construct was expressed on the T cell surface. Theresults in FIG. 13C show that the C2-BAR and OVA-BAR constructs weresuccessfully expressed on the transduced regulatory T cells.

This construct also can be also be used to transduce cytotoxic T cells.

Example 5—Functionality of BAR on Transduced Human CD4+ Regulatory TCells

To determine whether successfully expressed BAR constructs on thetransduced regulatory T cells can actually transmit signal and activatethe cells upon interaction with cognate B cell or antibody,proliferation response of OVA-BAR transduced human CD4+ T cells wastested. Human CD4+ regulatory T cells were transduced with an OVA-BARconstruct in a concentrated retroviral supernatant, labeled with cellproliferation dye eFlour450, and cultured with either cognate anti-OVAantibody or control IgG for 3 days in the presence of irradiated PBMC.Cell proliferation was analyzed by flow cytometry. The cells were gatedon live GFP+ cells. The results reported in FIG. 14 show thatOVA-BAR-transduced regulatory T cells proliferated specific to anti-OVAIgG but not to control IgG, which confirms that the BAR expressed on theregulatory T cells is functional.

Example 6—Long-Term Maintenance of Phenotype of Transduced HumanRegulatory T Cells

In order to obtain enough regulatory T cells for adoptive therapy,BAR-transduced regulatory T cells need to be expanded in vitro. Oneknown limitation facing human regulatory T cell therapy is that in vitroexpanded human regulatory T cells often quickly lose Foxp3 and/or Heliosexpression, and suppressive function. To confirm that BAR-transduced,long term in vitro-expanded human regulatory T cells retain theco-expression of Foxp3 and Helios, FACS analysis for Foxp3 and Heliosexpression was conducted on BAR-transduced regulatory T cells afterabout 3 weeks in vitro expansion. The results reported in FIG. 15 showthat BAR-transduced human regulatory T cells as described herein retainthe co-expression of Foxp3 and Helios after long term expansion.

Example 7—Generation of BAR-Transduced, In Vitro-Expanded HumanRegulatory T Cells

To transduce regulatory T cells with a BAR construct, human regulatory Tcells were first stimulated for two days. Stimulated cells weretransduced with concentrated retroviral supernatant. The transducedcells were maintained and rested for 8 days without additional stimuli.The GFP⁺ CD4 cells were isolated by FACS sorting, and used forre-stimulation and expansion. The quality of the transduced regulatory Tcells was checked by FACS, and the cells were harvest for experiment. Atimeline for generation of BAR-transduced regulatory T cells is shown inFIG. 16A.

Example 8—In vivo Suppression of Anti-FVIII Antibody Development by BARRegulatory T Cell Therapy in Naïve E16 Mice

A xenogeneic mouse model was used to validate the effectiveness ofBAR-transduced regulatory T cell therapy. As outlined in FIG. 16B, male6-10-week old E16 FVIII KO mice were divided into two groups. On dayone, five mice were intravenously injected with 1×10⁶ cells containingequal number of C2-BAR-transduced human Tregs and A2-BAR-transducedhuman regulatory T cells. As control, four mice received same number ofOVA-BAR-transduced human regulatory T cells. Both groups were activelyimmunized with FVIII in IFA adjuvant on day 1. The anti-FVIII antibodylevel was monitored by ELISA assay with a standard curve generated usinga mixture of anti-A2 and anti-C2 monoclonal antibodies. The resultsreported in FIG. 16C show that the group of mice treated with (C2+A2)BAR-transduced regulatory T cells had lower amounts of anti-FVIIIantibody compared to the control group. Thus, (C2+A2) BAR-transducedregulatory T cells essentially prevented anti-FVIII antibodydevelopment, confirming the effectiveness of BAR-transduced regulatory Tcell therapy in controlling unwanted immune response.

1. A method of targeting antigen-specific B cells, comprising exposing Bcells specific to a target antigen to regulatory T cells that express ontheir cell surface the target antigen or a domain thereof recognized bysaid antigen-specific B cells.
 2. The method of claim 1, wherein theregulatory T cells are transduced with a B-cell-targeting antibodyreceptor (BAR) construct comprising (i) an extracellular domaincomprising said target antigen or domain thereof and (ii) anintracellular signaling domain.
 3. The method of claim 2, wherein saidintracellular signaling domain comprises one or more signaling domainsof CD28-CD3ζ, 4-1BB, ICOS, and CTLA-4. 4.-5. (canceled)
 6. The method ofclaim 1, wherein the regulatory T cells are selected fromCD4⁺CD25^(hi)CD127^(lo)Foxp3⁺ regulatory T cells,CD4⁺CD25^(hi)CD127^(lo)Foxp3⁺Helios⁺ regulatory T cells,CD4⁺CD25^(hi)Foxp3⁺ regulatory T cells, and CD4⁺CD25^(hi)Foxp3⁺Helios⁺regulatory T cells. 7.-8. (canceled)
 9. The method of claim 3, whereinsaid intracellular signaling domain comprises one or more signalingdomains of CD28-CD3ζ, 4-1BB, and ICOS. 10.-11. (canceled)
 12. The methodof claim 9, wherein said T cells are selected from CD8+ T cells andnatural killer (NK) T cells.
 13. The method of claim 1, wherein themethod is effected in a patient suffering from or at risk of developingan undesirable immune response to said target antigen.
 14. The method ofclaim 13, wherein said patient is receiving a biotherapeutic treatmentfor a genetic disease selected from hemophilia or Pompe's, or isreceiving an allograft transplant. 15.-16. (canceled)
 17. The method ofclaim 13, wherein said target antigen is selected from human clottingfactor VIII, factor IX, and myelin basic protein (MBP).
 18. (canceled)19. The method of claim 13, wherein said undesirable immune response isassociated with an allergy, allergic response, or asthma and said targetantigen is an antigen associated with the allergy, allergic response, orasthma.
 20. The method of claim 1, wherein said target antigen or domainthereof recognized by said antigen-specific B cells is selected fromhuman clotting factor VIII, the C2 domain of human clotting factor VIII,the A2 domain of human clotting factor VIII, and the A2-C2 domain ofhuman clotting factor VIII. 21.-39. (canceled)